Approximately 10% of the population become hypersensitized (allergic) upon exposure to antigens from a variety of environmental sources. Those antigens that induce immediate and/or delayed types of hypersensitivity are known as allergens (King, T. P., (1976) Adv. Immunol., 23:77-105). Allergens can include products of grasses, trees, weeds, animal dander, insects, food, drugs, and chemicals. Genetic predisposition of an individual is believed to play a role in the development of immediate allergic responses (Young, R. P. et al., (1990) Clin. Sci., 79:19) such as atopy and anaphylaxis, whose symptoms include hay fever, asthma, and hives.
The antibodies involved in atopic allergy belong primarily to the IgE class of immunoglobulins. IgE binds to basophils, mast cells and dendritic cells via a specific, high-affinity receptor Fcxcex5RI (Kinet, J. P., (1990) Curr. Opin. Immunol., 2:499-505). Upon combination of an allergen acting as a ligand with its cognate receptor IgE, Fcxcex5RI bound to the IgE may be cross-linked on the cell surface, resulting in physiological manifestations of the IgExe2x80x94allergen interaction. These physiological effects include the release of, among other substances, histamine, serotonin, heparin, chemotactic factor(s) for eosinophilic leukocytes and/or leukotrienes C4, D4, and E4, which cause prolonged constriction of bronchial smooth muscle cells (Hood, L. E. et al., Immunology (2nd ed.), The Benjamin/Cumming Publishing Co., Inc. (1984)). Hence, the ultimate consequence of the interaction of an allergen with IgE is allergic symptoms triggered by the release of the aforementioned mediators. Such symptoms may be systemic or local in nature, depending on the route of entry of the antigen and the pattern of deposition of IgE on mast cells or basophils. Local manifestations generally occur on epithelial surfaces at the site of entry of the allergen. Systemic effects can induce anaphylaxis (anaphylactic shock) which results from IgE-basophil response to circulating (intravascular) antigen.
Studies with purified allergens have shown that about 80% of patients allergic to the mite Dermatophagoides pteronyssinus produce IgE reactive to Der p I and Der p II (Chapman M. D. et al., J. Immunol. (1980) 125:587-92; Lind P., J. Allergy Clin. Immunol. (1985) 76:753-61; Van der Zee J. S. et al., J. Allergy Clin. Immunol. (1988) 81:884-95). For about half the patients these specificities constitute 50% of the IgE antimite antibody. The allergen Der p III, recently identified as trypsin, (Stewart G. A. et al., Immunology (1992) 75:29-35) reacts with a similar or higher frequency (Stewart G. A. et al., supra; Ford S. A. et al., Clin. Exp. Allergy (1989) 20:27-31). However, in the only quantitative study reported to date, the investigators determined that the level of IgE binding to Der p III was considerably less than for Der p I. Electrophoretic techniques (Ford S. A. et al., supra; Bengtsson A. et al., Int. Arch. Allergy Appl. Immunol. (1986) 8:383-90; Lind P. et al., Scand. J. Immunol. (1983) 17:263-73; Tovey E. R. et al., J. Allergy Clin. Immunol. (1987) 2:93-102) have shown that most sera contain IgE which recognize other allergens.
The significance of the IgE reactivity to Der p III remains uncertain. The reactivity of this group of allergens has been reported to be as low as 16% using a fluid phase assay (Heymann et al., J Allergy Clin Immunol (1989) 83: 1055-1067) and as high as 100% using RAST assay (Stewart et al., Immunology (1992) 75: 29-35). Several others have reported IgE reactivity between 60-83% (Tovey et al., J Allergy Clin Immunol (1987) 79: 93-102; Thomas et al., Exp Appl Acarol (1992) 16:153-164; Yasueda et al., Clin Exp Allergy (1993) 23:384-390). The discrepancies in the frequency of IgE reactivity to the group III allergens may be attributable to either the differences in the purity of the allergen preparation studied or the differences in sensitivity of the assay techniques used. In order to determine the importance of particular specificities in the allergic reactions, there is a need for quantities of pure allergen, which would enable quantitative IgE binding tests and studies of the frequency and lymphokine profile of T cell responses to the allergen.
Many patients with sensitivity to house dust mite allergens are treated currently by administration of small, gradually increasing doses of house dust mite extracts. Use of these extracts has multiple drawbacks, including potential anaphylaxis during treatment and the necessity of continuing therapy, often for a period of several years to build up sufficient tolerance and significant diminution of clinical symptoms. The ability to substitute compositions of house dust mite allergen, Der p III, would overcome several of these drawbacks. Thus, a source of pure allergen that could be provided in quantity for use as a diagnostic or therapeutic reagent and therapeutic methods that would overcome the drawbacks associated with house dust mite extracts are highly desirable.
This invention provides isolated nucleic acids encoding peptides having at least one biological activity of Der p III, an allergen of the species Dermatophagoides pteronyssinus (house dust mite). A preferred nucleic acid is a cDNA having a nucleotide sequence shown in FIG. 1 (SEQ ID NO:1). The invention also pertains to peptides encoded by all or a portion of such cDNA (SEQ ID NO:1) and having at least one biological activity of Der p III. Also contemplated are isolated nucleic acids which hybridize under high stringency conditions (e.g., equivalent to 20-27xc2x0 C. below Tm and 1M NaCl) to a nucleic acid having a nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) or which encodes a peptide comprising all or a portion of an amino acid sequence of FIG. 1 (SEQ ID NO:2). Nucleic acids which encode peptides having an activity of Der p III and having at least 50% homology with a sequence shown in FIG. 1 (SEQ ID NO:2) are also featured. Peptides having a Der p III activity produced by recombinant expression of a nucleic acid of the invention, and peptides having a Der p III activity prepared by chemical synthesis are also featured by this invention. Preferred peptides have the ability to induce a T cell response which can include T cell stimulation (measured by, for example, T cell proliferation or cytokine secretion) or T cell non-responsiveness (i.e., contact with the peptide or a complex of the peptide with an MHC molecule of an antigen presenting cell induces the T cell to become unresponsive to stimulatory signals or incapable of proliferation). Other preferred peptides either apart from or in addition to the ability to induce a T cell response, have the ability to bind specific IgE of house dust mite-allergic subjects. Such peptides are useful in diagnosing sensitivity to house dust mites in a subject. Still other peptides, either apart from or in addition to the ability to induce a T cell response, have a significantly reduced or negligible ability to bind house dust mite-allergic IgE. Such peptides are particularly useful as therapeutic agents.
Other preferred peptides comprise an amino acid sequence shown in FIG. 1 (SEQ ID NO:2). In one embodiment, peptides having a Der p III activity and comprising a portion of the amino acid sequence of FIG. 1 (SEQ ID NO:2) are at least about 8-30 amino acids in length, preferably about 10-20 amino acids in length, and most preferably about 10-16 amino acids in length.
Another aspect of the invention features antibodies specifically reactive with peptides having a Der p III activity. Peptides having an activity of Der p III can be used in compositions suitable for pharmaceutical administration. For example, such compositions can be used in a manner similar to house dust mite extracts to treat or prevent allergic reactions to house dust mites in a subject. Nucleic acids of the invention and peptides having an activity of Der p III can also be used for diagnosing sensitivity in a subject to house dust mites.